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Abstract:

The present invention is directed to methods of treating eye disease. In
one embodiment, the method can comprise administering a taxane-cobalamin
bioconjugate or another taxane compound to a subject to treat the eye
disease. In one embodiment, the bioconjugate can be dissolved in an
aqueous solution prior to administration.

Claims:

1. A method of treating an eye disease, comprising administering a
bioconjugate to a subject to treat the eye disease, wherein the
bioconjugate comprises a taxane covalently bonded to a cobalamin.

2. The method of claim 1, wherein the taxane is covalently bonded to a
cobalt atom of the cobalamin.

3. The method of claim 1, wherein at least 80% of the bioconjugate is
dissolved in an aqueous solution prior to administration.

4. The method of claim 1, wherein the bioconjugate has a water solubility
of at least 50 mg/ml.

5. The method of claim 1, wherein the bioconjugate has a water solubility
of at least 100 mg/ml.

6. The method of claim 1, wherein the step of administering achieves
serum levels of about 0.1 ng/ml to about 20,000 ng/ml of the taxane in
the subject.

7. The method of claim 1, wherein the taxane portion of the bioconjugate
is administered at about 1 mg/kg/day to about 10 mg/kg/day.

8. The method of claim 1, wherein the taxane portion of the bioconjugate
is administered at about 2 mg/kg/day to about 6 mg/kg/day.

48. The method of claim 32, wherein the taxane compound includes a member
selected from the group consisting of paclitaxel and docetaxel,
derivatives thereof, and mixtures thereof.

49. The method of claim 32, wherein the administering is by ocular
delivery.

50. The method of claim 32, wherein the administering is by oral delivery
or by parenteral delivery.

Description:

BACKGROUND

[0001] The efficacy of certain drugs in treating disease is often
dependent on their toxicity, biological availability, or how readily an
effective amount of the drug can be delivered to a specific location in a
subject's body, particularly to a specific type of tissue or population
of cells. Therefore, methods and compositions that lower toxicity,
increase bioavailability, or facilitate drug targeting can be of
considerable value to the pharmaceutical and medicinal arts. One approach
to this need involves using molecules that have generally understood
transport mechanisms and which can be induced to release drugs in
site-specific fashion. Another approach to increasing bioavailability can
involve using molecules that broaden the options for formulating drugs,
so that the drugs can be administered in more effective dosage forms.

[0002] One such mechanism involves the use of cobalamin (Cbl). Cobalamin
is an essential biomolecule, the size of which prevents it from being
taken up from the intestine and into cells by simple diffusion, but
rather by facultative transport. Cobalamin must bind to a specific
protein, and the resulting complex is actively taken up through a
receptor-mediated transport mechanism. In the small intestine, cobalamin
binds to intrinsic factor (IF) secreted by the gastric lining. The Cbl-IF
complex binds to IF receptors on the lumenal surface of cells in the
ileum and is transcytosed across these cells into the bloodstream. Once
there, cobalamin binds to one of three transcobalamins (TCs) to
facilitate its uptake by cells. The receptor-mediated nature of cobalamin
uptake imparts a degree of cell-specificity to cobalamin metabolism, in
that cobalamin can be absorbed and metabolized by cells that present the
correct receptor(s).

[0003] Several patents have utilized cobalamin for various purposes. For
example, Grissom et al. has obtained several patents: U.S. Pat. Nos.
6,790,827; 6,777,237; and 6,776,976; using organocobalt complexes.
Russell-Jones et al. has also utilized cobalamin to increase uptake of
active agents, as described in a series of patents, including U.S. Pat.
Nos. 5,863,900; 6,159,502; and 5,449,720. In addition to this, research
and development for methods and compositions having increased
bioavailability of various pharmaceutical agents continue to be sought.

SUMMARY

[0004] It has been recognized that it would be advantageous to develop
compositions and methods for delivery of taxanes. Briefly, and in general
terms, the invention is directed to methods of treating an eye disease by
administering a taxane covalently bonded to a cobalamin as a
cobalamin-taxane bioconjugate to a subject. Alternatively, the method can
comprise administering a taxane compound to a subject to treat the eye
disease, wherein the taxane compound has a water solubility of at least
50 mg/ml. In one embodiment, paclitaxel is covalently bonded to the
cobalt atom of hydroxocobalamin, or more generally, one of the various
forms of vitamin B12. In another embodiment, a cobalamin-taxane
bioconjugate can be present in an aqueous solution, and can have a water
solubility of at least 50 mg/ml, or even over 100 mg/ml. Methods of
administering and/or treating an eye disease include administering a
cobalamin-taxane conjugate as an intra-ocular, oral, parenteral, or
dermal composition.

[0005] Additional features and advantages of the invention will be
apparent from the detailed description which follows, taken in
conjunction with the accompanying drawings, which together illustrate, by
way of example, features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Additional features and advantages of the invention will be
apparent from the detailed description which follows, taken in
conjunction with the accompanying drawings, which together illustrate, by
way of example, features of the invention; and, wherein:

[0007]FIG. 1 is a graph of various treatments after choroidal
neovascularization by laser burns of the eye at intervals of 7, 14, and
21 days; and

[0008]FIG. 2 is a bar graph of the mean lesion size (μm3)
corresponding to various treatments after choroidal neovascularization by
laser burns to the eye after 21 days.

[0009] Reference will now be made to the exemplary embodiments
illustrated, and specific language will be used herein to describe the
same. It will nevertheless be understood that no limitation of the scope
of the invention is is thereby intended.

DETAILED DESCRIPTION

[0010] Before the present invention is disclosed and described, it is to
be understood that this invention is not limited to the particular
structures, process steps, or materials disclosed herein, but is extended
to equivalents thereof as would be recognized by those ordinarily skilled
in the relevant arts. It should also be understood that terminology
employed herein is used for the purpose of describing particular
embodiments only and is not intended to be limiting.

[0011] In describing and claiming the present invention, the following
terminology will be used in accordance with the definitions set forth
below.

[0012] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and, "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a taxane" can include one or more of such taxanes,
and reference to "the cobalamin" can include reference to one or more
cobalamins.

[0013] As used herein, the terms "formulation" and "composition" can be
used interchangeably and refer to at least one pharmaceutically active
agent, such as a taxane covalently bonded to the cobalt atom of a
cobalamin with a covalent linkage. The terms "drug," "active agent,"
"bioactive agent," "pharmaceutically active agent," and "pharmaceutical,"
can also be used interchangeably to refer to an agent or compound that
has measurable specified or selected physiological activity when
administered to a subject in an effective amount. As used herein,
"carrier" or "inert carrier" refers to typical compounds or compositions
used to carry drugs, such as polymeric carriers, liquid carriers, or
other carrier vehicles with which a bioactive agent may be combined to
achieve a specific dosage form. As a general principle, carriers do not
substantially react with the bioactive agent in a manner that
substantially degrades or otherwise adversely affects the bioactive agent
or its therapeutic potential.

[0014] As used herein, "administration," and "administering" refer to the
manner in which a drug, formulation, or composition is introduced into
the body of a subject. Various art-known routes such as intra-ocular,
oral, parenteral, topical, transdermal, and transmucosal can be used for
administration. Thus, an intra-ocular administration can be achieved by
dissolving a bioconjugate in water and delivering directly to the eye;
e.g. via injection, eye drops, gels, or other topicals.

[0015] An oral administration can be achieved by swallowing, chewing,
dissolution via adsorption to a solid medium that can be delivered
orally, or sucking an oral dosage form comprising active agent(s).

[0016] Parenteral administration can be achieved by injecting a drug
composition intravenously, intra-arterially, intramuscularly,
intrathecally, or subcutaneously, etc. Topical administration may involve
applying directly to affected tissue, such as directly to the eye.
Transdermal administration can be accomplished by applying, pasting,
rolling, attaching, pouring, pressing, rubbing, etc., of a transdermal
preparation onto a skin surface. Transmucosal administration may be
accomplished by bringing the composition into contact with any accessible
mucous membrane for an amount of time sufficient to allow absorption of a
therapeutically effective amount of the composition. Examples of
transmucosal administration include inserting a suppository into the
rectum or vagina; placing a composition on the oral mucosa, such as
inside the cheek, on the tongue, or under the tongue; or inhaling a
vapor, mist, or aerosol into the nasal passage. These and additional
methods of administration are well known in the art.

[0017] The term "effective amount," refers to an amount of an ingredient
which, when included in a composition, is sufficient to achieve an
intended compositional or physiological effect. Thus, a "therapeutically
effective amount" refers to a non-lethal amount of an active agent
sufficient to achieve therapeutic results in treating a condition for
which the active agent is known or taught herein to be effective. Various
biological factors may affect the ability of a substance to perform its
intended task. Therefore, an "effective amount" or a "therapeutically
effective amount" may be dependent on such biological factors. Further,
while the achievement of therapeutic effects may be measured by a
physician or other qualified medical personnel using evaluations known in
the art, it is recognized that individual variation and response to
treatments may make the achievement of therapeutic effects a subjective
decision. In some instances, a "therapeutically effective amount" of a
drug can achieve a therapeutic effect that is measurable by the subject
receiving the drug. For example, in metronomic dosing, "the "therapeutic
effective amount" may increase or decrease during the therapeutic
treatment due to inherent genetic variation. The determination of an
effective amount is well within the ordinary skill in the art of
pharmaceutical, medicinal, and health sciences.

[0018] As used herein, "treat," "treatment," or "treating" refers to the
process or result of giving medical aid to a subject, where the medical
aid can counteract a malady, a symptom thereof, or other related adverse
physiological manifestation. Additionally, these terms can refer to the
administration or application of remedies to a patient or for a disease
or injury; such as a medicine or a therapy. Accordingly, the substance or
remedy so applied, such as the process of providing procedures or
applications, are intended to relieve illness or injury. As used herein,
"reduce" or "reducing" refers to the process of decreasing, diminishing,
or lessening, as in extent, amount, or degree of that which is reduced.
Additionally, the use of the term can include from any minimal decrease
to absolute abolishment of a physiological process or effect.

[0019] As used herein with respect to conditions of the eye, "disease"
refers to any condition of the eye that can result in diminished,
abnormal, or lost ocular function. This includes congenital disorders,
pathogenic disorders, and injury arising from physical, chemical, or
other trauma. This also includes trauma or other disturbance arising from
procedures conducted on the eye and intended to address such conditions.

[0020] As used herein, "subject" refers to an animal, such as a mammal,
that may benefit from the administration of a bioconjugate compound of
the present disclosure, including formulations or compositions that
include the compound.

[0021] As used herein, the term "taxane" generally refers to a class of
diterpenes produced by the plants of the genus Taxus (yews). This term
also includes those taxanes that have been artificially synthesized. For
example, this term includes paclitaxel and docetaxel, and derivatives
thereof.

[0022] As used herein, the term "cobalamin" refers to an organocobalt
complex having the essential structure shown below:

##STR00001##

as well as derivatives of this structure in which R may be --CH3
(methylcobalamin), --CN (cyanocobalamin), --OH (hydroxocobalamin),
--C10H12N5O3 (deoxyadenosylcobalamin), or synthetic
complexes that include a corrin ring and are recognized by cobalamin
transport proteins, receptors, and enzymes. The term also encompasses
inclusion of substituent groups on the corrin ring that do not eliminate
its binding to transport proteins. The term "organocobalt complex" refers
to an organic complex containing a cobalt atom having bound thereto 4-5
calcogens as part of a multiple unsaturated heterocyclic ring system,
particularly any such complex that includes a corrin ring.

[0023] The organocobalt molecule cobalamin is an essential biomolecule
with a stable metal-carbon bond. Among other things, cobalamin plays a
role in the folate-dependent synthesis of thymidine, an essential
building block of DNA. Because cobalamin is a large molecule, cellular
uptake of cobalamin is achieved by receptor-mediated endocytosis. The
density of receptors in a cell may be modulated in accordance with the
cell's need for cobalamin at a given time. For example, a cell may
upregulate its expression of cobalamin receptors during periods of high
demand for cobalamin. One such time is when the cell replicates its DNA
in preparation for mitosis or meiosis. One result of this facultative
upregulation is that cobalamin uptake will be higher in cell populations
undergoing rapid proliferation than in slower-growing cell populations.
This non-uniform uptake profile makes it possible to target delivery of a
bioactive agent to high-demand cell populations by linking the agent to
cobalamin.

[0024] Cobalamin is the most chemically complex of the vitamins. The core
structure of the cobalamin molecule is a corrin ring including four
pyrrole subunits, two of which are directly connected with the remainder
connected through a methylene group. Each pyrrole has a proprionamide
substituent that extends radially from the ring. At the center of the
ring is a cobalt atom in an octahedral environment that is coordinated to
the four corrin ring nitrogens, as well as the nitrogen of a
dimethylbenzimidazole group. The sixth coordination partner can vary as
previously discussed; represented by R in formula I. Six propionamide
groups extend from the outer edge of the ring, while a seventh links the
dimethylbenzimidazole group to the ring through a phosphate group and a
ribose group.

[0025] The term "vitamin B12" or "B12" has been generally used
in two different ways in the art. In a broad sense, it has been used
interchangeably with four common cobalamins: cyanocobalamin,
hydroxocobalamin, methylcobalamin, and adenosylcobalamin. In a more
specific way, this term refers to only one of these forms,
cyanocobalamin, which is the principal B12 form used for foods and
in nutritional supplements. For the purposes of this invention, this term
includes cyanocobalamin, hydroxocobalamin, methylcobalamin, and
adenosylcobalamin, unless the context dictates otherwise.

[0026] As used herein, the term "bioconjugate" refers to a molecule
containing a taxane covalently bonded to cobalamin, e.g., directly to the
cobalt atom or by some other linkage mechanism.

[0027] Exemplary of the bioconjugate function is the ability to solubilize
the taxane upon conjugation. As such, the present bioconjugates can have
water solubility allowing for direct dissolution of the bioconjugate in
water without the need for solubilization excipients. For example, a
taxane can be solubilized with CREMOPHOR®; however, such a solution
is toxic, which limits its therapeutic effectiveness and administration.
However, the present bioconjugates allow solubilization of taxanes in
water, or other aqueous solutions, without the need for further
excipients, which decreases toxicity and allows for intra-ocular
delivery.

[0028] Additionally, in one embodiment, the bioconjugate function can
serve as a targeted delivery system where the agent or compound to be
delivered may be conjugated or otherwise attached to cobalamin without
affecting the cobalamin's ability to bind to the appropriate receptor(s).
Therefore, it is often the case that the receptor-binding domain(s) of
the cobalamin are not modified. Likewise, for successful targeted
delivery, the agent or compound can be released from the cobalamin in a
therapeutically effective form and at the right location. Some event,
substance, or condition can be present in the targeted location that will
cause the agent to separate from the carrier. Successful methods of drug
targeting can involve agent-cobalamin linkages that are sensitive to
particular conditions or processes that are prevalent in the target
location.

[0029] As used herein, the term "covalent linkage" or "covalent bond"
refers to an atom or molecule which covalently or coordinate covalently
binds together two components. With regard to the present disclosure, a
covalent linkage is intended to include atoms and molecules which can be
used to covalently bind a taxane to cobalamin, such as through the
central cobalt atom in one embodiment. Though not excluded, in one
embodiment, the covalent linkage does not prevent the binding of
cobalamin to its transport proteins, either by sterically hindering
interaction between cobalamin and the protein, or by altering the binding
domain of cobalamin in such a way as to render it conformationally
incompatible with the protein. Likewise, the covalent linkage should not
act in these ways to significantly prevent the binding of the
cobalamin-transport protein complex with cobalamin receptors.

[0030] As used herein, the term "angiogenesis" or "angiogenic" refers to a
physiological process involving the growth of new blood vessels. The
growth of new blood vessels is an important natural process occurring in
the body, both in health and in disease. In regards to certain eye
diseases, the term "anti-angiogenic" refers to those compounds or agents
that inhibit the growth of new blood vessels, effectively cutting off the
existing blood supply of the disease(s). For example, such
anti-angiogenic compounds include, but are not limited to, bevacizumab,
suramin, sunitinib, thalidomide, tamoxifen, vatalinib, cilenigtide,
celecoxib, erlotinib, lenalidomide, ranibizumab, pegaptanib, sorafenib,
and mixtures thereof.

[0031] As used herein, the term "about" is used to provide flexibility to
a numerical range endpoint by providing that a given value may be "a
little above" or "a little below" the endpoint.

[0032] As used herein, a plurality of items, structural elements,
compositional elements, and/or materials may be presented in a common
list for convenience. However, these lists should be construed as though
each member of the list is individually identified as a separate and
unique member. Thus, no individual member of such list should be
construed as a de facto equivalent of any other member of the same list
solely based on their presentation in a common group without indications
to the contrary.

[0033] Concentrations, amounts, and other numerical data may be expressed
or presented herein in a range format. It is to be understood that such a
range format is used merely for convenience and brevity and thus should
be interpreted flexibly to include not only the numerical values
explicitly recited as the limits of the range, but also to include all
the individual numerical values or sub-ranges encompassed within that
range as if each numerical value and sub-range is explicitly recited. As
an illustration, a numerical range of "about 1 micron to about 5 microns"
should be interpreted to include not only the explicitly recited values
of about 1 micron to about 5 microns, but also include individual values
and sub-ranges within the indicated range. Thus, included in this
numerical range are individual values such as 2, 3.5, and 4 and
sub-ranges such as 1-3, 2-4, and 3-5, etc. This same principle applies to
ranges reciting only one numerical value. Furthermore, such an
interpretation should apply regardless of the breadth of the range or the
characteristics being described.

[0034] In accordance with these definitions, the present invention
provides methods of treating eye diseases by administering a composition
to a subject where the composition includes a taxane or derivative
covalently bound to cobalamin. Alternatively, a method of treating an eye
disease can comprise administering a taxane compound to a subject to
treat the eye disease, wherein the taxane compound has a water solubility
of at least 50 mg/ml. It is noted that when discussing a cobalamin-taxane
bioconjugate or taxane compound or a method of administering such a
composition, each of these discussions can be considered applicable to
other embodiments describe herein, whether or not they are explicitly
discussed in the context of that embodiment. Thus, for example, in
discussing taxanes bioconjugates or taxane compounds, the details of the
methods can be used interchangeably.

[0035] In one embodiment, the bioconjugate can comprise a taxane
covalently bonded to a cobalamin. In another embodiment, the taxane can
be covalently bonded to a central cobalt atom of the cobalamin, and in
another embodiment, the bioconjugate can be present as a solubilized
compound in an aqueous solution. The step of administering can be
accomplished by various methods as are known in the art.

[0036] In one embodiment, the step of administering can be by intra-ocular
administration or delivery. In another embodiment, the step of
administering can be by oral administration or delivery. In yet another
embodiment, the step of administering can be by parenteral administration
or delivery. In still yet another embodiment, the step of administering
can be by topical delivery to the tissue site, or by dermal or mucosal
administration or delivery.

[0038] In one embodiment, the present bioconjugates can treat age related
macular degeneration (AMD). Specifically, AMD general can be described in
two forms: dry and wet. Dry is most common and does not have
neovascularization. However, dry AMD can lead to wet AMD. Wet AMD has
neovascularization which is the development of abnormal leaky blood
vessels in the macular of the eye. This can result in blindness and/or
very impaired vision. Wet AMD is an angiogenic process, i.e., it is the
development of new blood vessels that are weak and leaky. These occur in
the macula and as a result, can also lead to bleeding in the eyes from
the vessels leaking blood. As such, the present bioconjugates can be used
for the treatment of AMD, as a result of their anti-angiogenic benefits,
as further described herein. Additionally, in another embodiment, the
present bioconjugates can treat diabetic retinopathy (both
non-proliferative and proliferative) as such diseases are known to have
abnormal blood vessel growth.

[0039] The present eye diseases can benefit from administration of the
present bioconjugates, e.g., B12-paclitaxel, since such
bioconjugates are water soluble allowing for direct solubilization in
water, or other aqueous solutions, without the to need for toxic
solubilizing excipients, e.g., CREMOPHOR®. Additionally, the
bioconjugates can be nontoxic in the eye at doses up to 85 μg/2 μL.

[0040] Generally, attaching the taxane to the cobalt atom of cobalamin
more closely approximates the binding arrangement seen in stable,
biologically active forms of cobalamin, such as adenosylcobalamin. It has
been recognized that the attachment of a taxane to the cobalt atom of a
cobalamin can significantly increase the water solubility of the taxane
as a cobalamin-taxane bioconjugate. Thus, such an arrangement can be
beneficial for treating eye disease, though other forms of such
bioconjugates can also be used when solubility is not the objective,
e.g., emulsions, microemulsions, liposomes, etc.

[0041] Generally, taxanes are insoluble in water. For example, paclitaxel
has a water solubility of less than 0.004 mg/ml. However, when conjugated
to a cobalt atom of a cobalamin, as shown in the following structure and
described herein, a cobalamin-paclitaxel bioconjugate can have water
solubility of over 100 mg/ml, though lesser degrees of solubility with
certain molecules can also be effective for treatment as well. For
example, in one embodiment, a cobalamin-taxane bioconjugate can have a
water solubility of at least 0.5 mg/ml. In another embodiment, a
cobalamin-taxane bioconjugate can have a water solubility of at least 10
mg/ml. In yet another embodiment, the water solubility can be at least 50
mg/ml. In still yet another embodiment, the water solubility can be at
least 100 mg/ml. In one embodiment, at least 80% of the bioconjugate can
be dissolved in an aqueous solution prior to administration. It is noted
that the cobalamin-taxane bioconjugates provided herein can be orally
administered to a subject or can be delivered directly to the eye, or by
some other effective administration route. In one embodiment, paclitaxel
can be covalently bonded to the cobalt atom of a hydroxocobalamin.
Specifically, the cobalamin-taxane bioconjugate can be a
cobalamin-paclitaxel bioconjugate having the following structure:

##STR00002##

[0042] Alternatively, the cobalamin-taxane bioconjugate can be a
cobalamin-docetaxel bioconjugate having the following structure:

##STR00003##

In each of the two above structures as well as in other similar
embodiments, it is understood that although the Cl-counter ion is
shown, other similar pharmaceutically acceptable counter ions can
alternatively be used.

[0043] The cobalamin-taxane bioconjugates can have a water solubility
several orders of magnitude higher than unconjugated taxanes. In one
embodiment, the cobalamin-taxane bioconjugate can have at least a 10-fold
increase in water solubility compared to the unconjugated taxane. In
another embodiment, the increase can be at least 100-fold. In yet another
embodiment, the increase can be at least 1000-fold.

[0044] Additionally, it has been recognized that the cobalamin-taxane
bioconjugates disclosed herein can have increased bioavailability in a
subject. Bioavailability of a compound can be dependent on P-Glycoprotein
(P-gp), an ATP-dependent drug pump, which can transport a broad range of
hydrophobic compounds out of a cell. This can lead to the phenomenon of
multi-drug resistance. Expression of P-gp can be quite variable in
humans. Generally, the highest levels can be found in the apical
membranes of the blood-brain/testes barrier, intestines, liver, and
kidney. Over-expression in patients can undermine treatment as the drug
is pumped out via this pump. P-gp can also affect the penetration of the
drug to solid tumors or other maladies. P-gp has been shown to affect the
ability of taxanes, such as paclitaxel or docetaxel, to enter the cells
and become bioavailable. Therefore, the bioconjugates of the present
invention can be structurally different as to bypass the P-gp pathway
leading to increased bioavailability of the bioconjugate. Additionally,
cobalamin bioconjugates can use a facultative transport mechanism, which
would also bypass the P-gp pathway leading to increased bioavailability.

[0045] The present disclosure also relates to solubilization and drug
delivery of taxanes and their derivatives for the treatment of the eye
via a cobalamin-taxane bioconjugate, e.g., oral, parenteral, topical,
ocular, etc. In addition, it is noted that there may be an inherent
targeting effect via the cobalamin molecule. When introduced into the
bloodstream or gastrointestinal tract of a subject, such a bioconjugate
can take advantage of existing systems for absorption, transport, and
binding of cobalamin. In this way, the taxane can be transported to cells
that bear receptors for cobalamin and be taken up by those cells. As
noted above, some cells or cell populations in a given subject can
utilize cobalamin more heavily at a given time than other cells;
consequently expression of cobalamin receptors is upregulated in such
cells at those times. Thus, when the bioconjugate is administered to a
subject, more of the taxane can be taken up by these cells than by other
cells. Thus, the present invention provides a method for concentrating a
taxane to sites where cells are utilizing cobalamin heavily. Increased
demand for cobalamin is associated with, among other things, rapid
cellular proliferation. Therefore, the present invention can be used to
concentrate taxanes in neoplastic cells in a subject suffering from a
proliferative disease.

[0046] The taxane can be covalently bonded to the cobalt atom directly or
through a covalent linkage. The linkage serves as a connection between
the cobalamin and the taxane, and can serve to achieve a desired distance
between these two components, while preferably not negatively affecting
the binding of the bioconjugate to proteins involved in cobalamin
metabolism. In particular, the linkage can include an ester linkage.
Alternatively or additionally, the linkage can include a quaternary
amine. In another alternative embodiment, the linkage could be a
hydrazone linkage. The bioconjugate of the present invention can also
include a linkage comprising a polymethylene, carbonate, ether, acetal,
or any combination of these units.

[0047] Though specific structures and discussions are provided above, it
is noted that in a more general embodiment, the cobalamin-taxane
bioconjugate can be linked as follows:

##STR00004##

where Y is any alkyl containing 1 to 4 carbons; and X is an optionally
substituted, saturated, branched, or linear, C1-50 alkylene,
cycloalkylene or aromatic group, optionally with one or more carbons
within the chain being replaced with, N, O or S, and wherein the optional
substituents are selected from carbonyl, carboxy, hydroxyl, amino and
other groups. The "Acid" can be any organic or inorganic acid, preferably
having the ability to form pharmaceutically acceptable salts. Other
linkages that will serve the functions described above will be known to
those having skill in the art, and are encompassed by the present
invention.

[0048] Such a linkage can serve as a target for an enzyme that will cleave
the linkage, releasing the taxane from the cobalamin. Such an enzyme can
be present in the subject's bloodstream and thereby release the taxane
into the general circulation, or it can be localized specifically to a
site or cell type that is the intended target for delivery of the taxane.
Alternatively, the linkage can be of a type that will cleave or degrade
when exposed to a certain environment or, particularly, a characteristic
of that environment such as a certain pH range or range of temperatures.
The linkage can be of a "self-destructing" type, i.e. it will be consumed
in the process of cleavage, so that said cleavage will yield only the
original cobalamin and the taxane molecules absent any remaining large
sections of the linkage. Those having skill in the art will recognize
other release mechanisms derived from various linkages that can be used
in accordance with the present invention.

[0049] Again, though specific compounds are shown by way of example, it is
understood that many different combinations of taxanes and cobalamin can
be prepared in accordance with embodiments of the present disclosure. For
example, the taxane for use can be selected from the group consisting of
paclitaxel and docetaxel, derivatives thereof, and mixtures thereof. In
one embodiment, the taxane can be paclitaxel. In another embodiment, the
taxane can be docetaxel. The cobalamin can be selected from the group
consisting of cyanocobalamin including anilide, ethylamide,
proprionamide, monocarboxylic, dicarboxylic, and tricarboxylic acid
derivatives thereof; hydroxycobalamin including anilide, ethylamide,
proprionamide, monocarboxylic, dicarboxylic, and tricarboxylic acid
derivatives thereof; methylcobalamin including anilide, ethylamide,
proprionamide, monocarboxylic, dicarboxylic, and tricarboxylic acid
derivatives thereof; adenosylcobalamin including anilide, ethylamide,
proprionamide, monocarboxylic, dicarboxylic, and tricarboxylic acid
derivatives thereof; aquocobalamin; cyanocobalamin carbanalide;
desdimethyl cobalamin; monoethylamide cobalamin; methlyamide cobalamin;
5'-deoxyadenosylcobalamin; cobamamide derivatives; chlorocobalamin;
sulfitocobalamin; nitrocobalamin; thiocyanatocobalamin; benzimidazole
derivatives including 5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole,
trimethylbenzimidazole, as well as adenosylcyanocobalamin; cobalamin
lactone; cobalamin lactam; 5-o-methylbenzylcobalamin; derivatives
thereof; mixtures thereof; and analogues thereof wherein the cobalt is
replaced by another metal. In one embodiment, the cobalamin can be one of
the vitamin B12 types of cobalamin, and in one specific embodiment,
hydroxocobalamin.

[0050] The compounds of the present invention can be administered as
pharmaceutical compositions in treating various eye diseases.
Notwithstanding the ability to solubilize taxanes without the need for
solubilizing excipients and/or additives, such a composition can further
comprise one or more excipients, including binders, fillers, lubricants,
disintegrants, flavoring agents, coloring agents, sweeteners, thickeners,
coatings, and combinations thereof. The composition of the present
invention can be formulated into a number of dosage forms including
syrups, elixirs, solutions, suspensions, emulsions, capsules, tablets,
lozenges, and suppositories. Differing administration regimens will call
for different dosage forms, depending on factors such as the subject's
age, medical condition, level of need for treatment, as well as the
desired time course of therapeutic effect. Those having skill in the art
will recognize that various classes of excipients can each provide
different characteristics to a pharmaceutical composition and that they
can be combined in certain ways in accordance with the present invention
to achieve an appropriate dosage form. The present invention provides
compounds that can be administered to a subject intra-ocularly, orally,
dermally, or parenterally.

[0051] One aspect of the present invention is that administering the
bioconjugate can be more effective in treating an eye disease than
administering the taxane and the cobalamin as separate molecules. In
light of the fact that taxanes alone can provide anti-angiogenic effects,
the present invention provides cobalamin-taxane bioconjugates as
anti-angiogenic compounds for treating various eye diseases. The amount
of taxane to cobalamin can generally be equal, e.g., the taxane to
cobalamin molar ratio can about 1:1. However, the composition can have an
excess of cobalamin or taxane that is not covalently bonded. In one
embodiment, a composition can have a cobalamin to cobalamin-taxane
bioconjugate molar ratio from about 1.2:1 to about 10:1. Additionally,
the bioconjugate can further include additional anti-angiogenic
compounds. Such additional anti-angiogenic compounds include, but are not
limited to, bevacizumab, suramin, sunitinib, thalidomide, tamoxifen,
vatalinib, cilenigtide, celecoxib, erlotinib, lenalidomide, ranibizumab,
pegaptanib, sorafenib, and mixtures thereof.

[0052] As previously discussed, the bioconjugates of the present invention
are readily soluble in water and can be administered to a subject having
various eye diseases. As such, the administering can be therapeutically
effective while providing low serum levels in the patient, enabling
effective treatments having no or very little toxicity. Specifically, the
serum levels can be less than 0.01 ng/ml. In another embodiment, the
serum levels can be less than 0.001 ng/ml. The taxane of the bioconjugate
can be administered at, or equivalent to, about 0.001 μg/day to about
10 μg/day.

[0053] As cobalamin receptors are highly upregulated in rapidly
proliferating cells as dividing cells require cobalamin for thymidine
synthesis in DNA replication. This makes cobalamin a useful vehicle to
preferentially deliver drugs to proliferating cells. In one embodiment,
administering the bioconjugates of the present invention can be used to
achieve serum levels in a subject of about 0.1 ng/ml to about 20,000
ng/ml. Further, the taxanes of the cobalamin-taxane bioconjugates of the
present invention can be administered at about 1 mg/kg/day to about 10
mg/kg/day. In one embodiment, the rate can be about 2 mg/kg/day to about
6 mg/kg/day.

[0054] It is to be understood that the above-described arrangements are
only illustrative of the application of the principles of the present
invention. Numerous modifications and alternative arrangements may be
devised by those skilled in the art without departing from the spirit and
scope of the present invention and the appended claims are intended to
cover such modifications and arrangements. Thus, while the present
invention has been described above with particularity and detail in
connection with what is presently deemed to be the most practical and
preferred embodiments of the invention, it will be apparent to those of
ordinary skill in the art that numerous modifications, including, but not
limited to, variations in size, materials, shape, form, function and
manner of operation, assembly and use may be made without departing from
the principles and concepts set forth herein.

EXAMPLES

[0055] The following provides examples of taxane bioconjugates in
accordance with the compositions and methods previously disclosed.
Additionally, some of the examples include studies performed showing the
effects of oral taxanes on animals in accordance with embodiments of the
present invention.

Example 1

Preparation of Cobalamin-Paclitaxel Bioconjugate

[0056] A cobalamin-paclitaxel bioconjugate was prepared using the
following reaction schematic:

[0061] Hydroxocobalamin acetate (0.5 g, 0.355 mmol) is dissolved in DI
H2O (25 ml), and N-methyl-3-chloropropylamine (0.108 g, 0.751 mmol)
and NH4Cl (0.195 mg, 3.63 mmol) is added to the solution. The
solution is degassed by bubbling with N2 for 30 min. Then, 0.238 g
Zn dust (3.63 mmol; <10 micron) is added in one portion. All the
starting material is consumed after the reaction is stirred under N2
for 3.5 h. The reaction mixture is then filtered with Whatman No. 42
filter paper to remove Zn. The filtrate is loaded on a Waters C18
Sep-Pak® cartridge (10 g of C18 sorbant) that is pre-washed by
washing with 60 ml of methanol followed with 100 ml of water. All salts
are removed from the cartridge with 100 ml of water and the product is
eluted with CH3OH--H2O (9:1) and concentrated to dry. The
residue is resuspended in 4 ml of methanol and precipitated in 100 ml of
1:1 (V/V) CH2Cl2/anhydrous Et2O. The red solid is filtered
and washed with acetone (20 ml) and ether (20 ml), affording 0.482 g
(yield 94.6%, purity 98%) of product.

Preparation of (3) Cbl-(CH2)3N(CH3)CH2COO-2'-PTX:

[0062] A solution of compound (1) (0.743 g, 0.799 mmol, 1.0 eq), compound
(2) (1.976 g, 1.374 mmol, 1.72 eq), and DIEA (0.24 ml, 1.374 mmol, 1.72
eq) in DMSO (48 ml) is stirred at room temperature for 3 days. HPLC is
employed to confirm consumption of compound 1. The reaction mixture is
added to stirring CH2Cl2/ether (1:2, 450 ml). The resulting
precipitate is collected, washed with CH2Cl2 (20 ml×3)
and ether (20 ml×3), and air-dried. The crude product is diluted
with 0.01 N HCl (200 ml) and applied to a C18 reverse phase 43 g
column which is pre-washed sequentially with 7 volumes of methanol and
water. The column is first washed with water (50 ml) and eluted with
5-40% B in buffer A (200 ml each with 5% increment). The fractions are
checked for purity by HPLC. The desired fractions are combined, diluted
with one volume of water, and adsorbed onto a Waters C18 Sep-Pak®
cartridge (10 g, P/N WAT043350, pre-washed sequentially with 3 volumes of
methanol and water). The product is washed with water (20 ml×3),
0.01 M HCl (20 ml×3), water (20 ml×3) and eluted off the
cartridge with 9:1 acetonitrile/water (50 ml). The organic solvent is
removed with a rotary evaporator. The residue is dissolved in 0.01 N
hydrochloride solution (40 ml, with the aid of a few drops of 0.1 N
hydrochloride solution), filtered by 0.45 μm NYLON membrane filter,
and lyophilized. 780 mg (41.9%) of red powder is obtained. ES(+)-MS:
1148.9 [(M+H)2+], 1329.9 (Cbl.sup.+), 665.7 [(Cbl+H)2+], 971.6
[(Cbl-359).sup.+], 359.1 (fragment from the breakdown of C--OP(O) bond).

[0063] The resultant compound has the following structure:

##STR00008##

Example 2

Preparation of Cobalamin-Docetaxel Bioconjugate

[0064] Similar procedures are followed as outlined in Example 1, but with
docetaxel as the principal taxane, resulting in the following structure:

##STR00009##

Example 3

Cobalamin-Paclitaxel Bioconjugate Dose Study

[0065] A group of 6 mice are administered various dosages of the
cobalamin-paclitaxel bioconjugate prepared in accordance with Example 1
over a 28-day period. The effects on counts of viable circulating
endothelial cell precursors and white blood cells are measured after 28
days. Corresponding amounts of the cobalamin-paclitaxel bioconjugate,
viable circulating endothelial cell precursors (CEPS), and white blood
cells are presented in the Table 1:

As can be seen from Table 1, administration of the cobalamin-paclitaxel
bioconjugate has an anti-angiogenic effect (marked decrease in viable
CEPS) at each dose. However, the most effective dose is not proportional
to the amount of paclitaxel administered. In fact, the most effective
dose in this particular study is about 2 mg/kg. Furthermore, the absence
of a decrease in the white blood cell count shows that such a dosage is
less toxic to the mouse (no neutropenia).

[0066] A Matrigel® plug perfusion in vivo assay is performed to
determine the anti-angiogenic efficacy of the cobalamin-paclitaxel
bioconjugate (Cob-Pac) of Example 1. The assay uses Matrigel®, a
gelatinous protein mixture secreted by mouse tumor cells (BD Biosciences,
San Jose, Calif.), to duplicate tissue environments. Matrigel® is
liquid at room temperature, but when injected into the animal, forms a
solid plug. If a growth vessel stimulant such as basic fibroblast growth
factor (bFGF) is mixed with the Matrigel®, the bFGF stimulates the
formation of new blood vessel in the plug, which can be monitored in the
animal via fluorescence techniques. In the current study, Matrigel®
is injected either alone or with bFGF subcutaneously into mice. Then, as
indicated in Table 2, groups of mice are either treated by oral gavage
with the cobalamin-paclitaxel conjugate or in the last group with the
mouse anti-VEGF receptor antibody, DC101. The results are shown in Table
2:

[0067] Such results indicate that the addition of bFGF stimulates the
growth of blood vessels on the Matrigel® assay as indicated by the
fluorescence ratio in the Matrigel® plus bFGF. The addition of
cobalamin-paclitaxel bioconjugate inhibits the growth of new blood
vessels in each instance shown. However, the greatest effect is seen at
the 2 mg/kg (expressed in paclitaxel units) and 6 mg/kg (expressed in
paclitaxel units) doses. The cobalamin-paclitaxel bioconjugate can
provide better performance than that of DC101, an effective rodent
specific anti-angiogenic compound that is well known in the art.

Example 5

Choroidal Neovascularization Model

[0068] Groups of 8 rats/dosage group or vehicle are neovascularized by
laser burns on the eye. Afterwards, the eye is immediately treated with a
cobalamin-paclitaxel bioconjugate prepared in accordance with Example 1
at a dose of 1.5 μg/2 μL, 5.0 μg/2 μL, and 15 μg/2 μL
(indicated as B.C. in FIG. 1). The treatment regimen also includes a
vehicle and Kenacort Retard® (4% triamcinolone acetonide), as a
positive control. Each treatment is scored at 7, 14, and 21 days
post-treatment by infusing the eye with fluorescein and scoring the
leakage using angiography. A score of 0 indicates no leakage while a
score of 3 indicates severe leakage. The results of the test are shown in
FIG. 1 as the percentage of mice scoring 3.

[0069] As can be seen in FIG. 1, after 7 days, the present bioconjugate in
the intermediate and high doses show anti-angiogenic results. After 14
days, the high dose still provides anti-angiogenic benefit. Such results
show that the compound of Example 1 can be effective for preventing new
BV growth in the eye.

Example 6

Lesion Study

[0070] In another study, flat mount evaluation of the eyes can be carried
out at the end of the study because angiography may not provide a full
evaluation of the effect of the drug. At the end of the study, the eyes
are removed, histologically processed and all lesions can be seen
(including those that can not be detected by angiography). Such an
evaluation can be used as a better measure of the choroidal
neovascularization model.

[0071] Groups of 8 rats/dosage group are neovascularized by laser burns,
followed by immediate treatment of the eye with a cobalamin-paclitaxel
bioconjugate prepared in accordance with Example 1. Dosages used are 1.5
μg/2 μL, 5.0 μg/2 μL, and 15 μg/2 μL. The treatments
also include a vehicle and Kenacort Retard® (4% triamcinolone
acetonide). After 21 days, the eyes are removed, histologically
processed, and the lesion size scored in μm3. FIG. 2 shows the
results of the treatments.

[0072] As illustrated in FIG. 2, the number of blood vessel lesions
decrease in a concentration dependent manner after treatment using the
B12-paclitaxel bioconjugate of Example 1, indicating dose dependent
inhibition of blood vessel growth. As such, the present study provides a
more detailed analysis than the angiography results from Example 5. The
present study demonstrates that both the high and medium concentrations
of B12-paclitaxel can be efficacious in inhibiting new blood vessel
growth.

[0073] While the invention has been described with reference to certain
preferred embodiments, those skilled in the art will appreciate that
various modifications, changes, omissions, and substitutions can be made
without departing from the spirit of the invention. It is therefore
intended that the invention be limited only by the scope of the appended
claims.